In various situations and applications of e.g. medical care, food handling and food storing it is very important that devices and products that are used are kept free from growth or proliferation of microorganisms. Not the least is this extremely important when it comes to medical devices that are brought into contact with patients in a hospital, since contaminated devices may participate in spreading of disease and microorganisms in a way that may severely affect the health of many patients.
It would for instance be very advantageous if the devices and products that come into contact with potentially harmful microorganisms had the capacity to inhibit or kill bacteria and/or other microorganisms, such as virus and fungi, in order to prevent the spreading of diseases.
The objective is to prevent the initial colonization that subsequently may develop into a biofilm. The initial phase of colonization may be suppressed by either inhibition or the killing of micro-organisms.
In order to obtain such a protection it is known to provide a surface in a device with metal ions, such as ions of the elements Ag and Ni. Often Ag is applied as an alloy with the purpose to release the Ag+-ions at a suitable rate to the environment, thereby preventing the accumulation of microorganisms.
However, one problem with this solution is the adhesion of the metal or alloy to the surface in question. Also, the antimicrobial effect is not easily controlled, and Her the metal ion coated surface may have cytotoxic effects.
U.S. Pat. No. 6,475,434 discloses a biofilm penetrating composition for removal of biofilms formed and constituted by infectious microorganisms as well as for coating medical devices in order to prevent formation of such biofilms. The composition comprises cysteine and analogues or derivatives thereof to be selected as one of the components. The role of the cysteine or cysteine related component is unclear and notably for coating applications they are used in combination with known antimicrobial agents such as rifamycines, tetracyclines and penicillines. Notably, as shown in examples 2 and 3 in U.S. Pat. No. 6,475,434, the only cysteine component tested (which is N-Acetyl Cysteine) has no effect unless combined with the antibiotics tested. Furthermore for biofilm protection all components are applied by impregnation of the device or mixing with the device material during manufacturing. The components then become physically bound by adhesion and penetration into the device material, which means that their function to a large extent occurs upon their release to the environment. Particularly in medical application such concepts require strict control of the balance between antimicrobial, cytotoxic and immunogenic effects. Since diffusion is time and temperature dependent, storage and durability of the coated devices also become matters of serious concern.
It has been described by Olofsson et al. (Applied and Environmental Microbiology, August 2003; 69(8), 4814-4822) that N-Acetyl Cysteine can affect bacterial growth in solution. Other effects of N-Acetyl Cysteine were to diminish the adhesion of multi-species bacteria onto stainless steel surfaces or facilitating the detachment of a biofilm on stainless steel surfaces.
A primary purpose of the present invention is to provide an antimicrobial agent having the capability to prevent or at least substantially reduce the accumulation and/or adhesion of individual microorganisms on the surface of a device in a sable and long-term manner.
This purpose is fulfiled by the inventors in a first aspect of the invention, referring to an antimicrobial agent cowing a subs with a covalently bound cysteine compound.
In particular, the invention provides an antimicrobial agent wherein the cysteine compound is bound through an S—S bridge via a spacer molecule to the substrate. The spacer comprises a carbon chain, optionally interrupted by one or more heteroatoms, e.g. O, S, N, P or Si, and the chain is optionally substituted with one or more alkyl groups, preferably lower alkyl groups with 1-6 carbon atoms, hydroxyl groups or alkoxy groups. In the examples given below the cysteine compound is bound in a terminal position of the spacer via an S—S bridge, which is a preferred embodiment of the invention. However, also other positions in the spacer chain are possible as long as the cysteine function is exposed to the environment.
According to a preferred embodiment of the invention the cysteine-containing ligand bound to a substrate has the general formula:R1—X-L-S—S—(cysteine component),  (1)wherein
R1 is a soluble or insoluble substrate; e.g. a solid surface or a soluble organic molecule or polymer.
X is a linking group from the coupling reaction between the substrate and L.
L is a spacer molecule selected from the group comprising (CH2)m where m is 1-20, preferably 1-12, 1-8 or 1-6; (CH2CH2O)n(CH2)p or (CH(CH3)CH2O)n(CH2)p where n is 1-1000, preferably 1-100 or 3-50, and p is 1-20, preferably 1-12, 1-10 or 1-6. The (CH2)p segment is bound to the disulphide bridge but may optionally also occur between the (CH2CH2O)n and/or (CH(CH3)CH2O)n segments in a block co-polymer;
Cysteine component is herein referred to as the residue of a cysteine compound comprising cysteine, a cysteine analogue or cysteine derivative providing antimicrobial act which is substantially the same or on a comparable level to that confer by cysteine It has been noticed that an embodiment of the invention where the cysteine compound is bound via an S—S bridge comprising one S from the thiol group of cysteine compound and one S from the spacer molecule is of special importance, at least in some applications, due to its superior antimicrobial activity.
Accordingly, an antimicrobial agent is provided that is covalently attached to the of the device, and which, due to the surprisingly advantageous effects of covalently bound cysteine, has a high and long-term effect; see the examples below, on individual microorganisms, thereby preventing or substantially inhibiting the adhesion and accumulation of individual microorganisms. Thus, the present invention offers a huge potential for all applications wherein it is desirable that a surface or substrate will exhibit antimicrobial/antibacterial properties. A flier and essential advantage of the invention, is that it has been shown by the inventors that the agent of the invention appears to lack cytotoxic effects, which makes it usable in many different applications.
In one aspect of the present invention various devices that are desirable to keep free from accumulation and/or adhesion of microorganisms are coated completely or partially with an antimicrobial agent according to the invention.
In a further aspect, the present invention refers to the use of an antimicrobial agent of the invention for preventing growth and/or proliferation of microorganisms on a substrate and/or a surface of a device.
In contrast to U.S. Pat. No. 6,475,434, the present invention provides a method to have the cysteine or cysteine related component covalently bound to a substrate. A major use for the invention is to provide an antimicrobial coating to a solid device. By this concept the antimicrobial agents are permanently attached to the surface and the antimicrobial effect occurs upon surface contact rather than from reaction with released agents which will largely decrease the risk of adverse effects in a biological environment. This is a major difference compared to the prior art methods where cysteine is provided as such a release agent. Also by covalent attachment the surface can be made more specified in terms of surface concentration and chemical structure. Thus not only the surface bound cysteine or cysteine related component itself but at least in some applications also the disulphide bond by which it is linked to the surface is one of the inventive features of the present concept with regard to antimicrobial effect. Furthermore, the covalent attachment provides a surface, which in situ as well as in storage is superior in consistency and durability compared to surfaces where diffusion and leakage of the active agents are of major concern.
“An antimicrobial agent” comprises a substrate which has been modified to exhibit a covalently bound cysteine component and has the effect of preventing, or at least substantially preventing accumulation, growth and/or proliferation of at least one specific microorganism. This effect can e.g. be observed by methods known in the art, e.g. by the methods used in the example section of this disclosure.
A “cysteine component” comprises the residue of cysteine, a cysteine analogue or cysteine derivative having antimicrobial effect, e.g. homocysteine or N-substituted cysteines such as N-acetyl-L-cysteine and N-alkylated cysteines.
“Substrate” (R1) comprises any article, device, molecule or polymer, soluble or insoluble that can be functionalized to obtain antimicrobial properties by binding a cysteine component. Of special interest are solid articles like medical devices to be used inside or in contact with the human or animal body, in particular sensitive tissues and body fluids. This list of potential applications is extensive, see further below, and includes implants, tubings drainage catheters, etc to be used in e.g. extracorporeal applications, drainage (e.g. ear or hydrocephalus), dialysis, contact lenses, intraocular lenses, artificial skins, dialysis equipments, heart and lung machines, suture materials, wound care devices, dental products, parenteral administration, drug delivery, stents, pumps (e.g. for insulin), hearing aid devices, syringes, suture materials, pacemakers, etc.
“Preventing” or “inhibiting” comprises the capacity to stop or substantially reduce growth and/or proliferation and/or accumulation and/or substantially reduce viability of microorganisms at a position where the agent of the invention is present
The main potential of the present invention is to offer the possibility to provide an antimicrobial surface on a solid device, which potentially comes in contact with microorganisms, and which is desirable to keep free from accumulation and/or proliferation of microorganisms and/or serve as a reservoir for viable microorganisms. The great number of devices to be used in medical as well as food-handling applications where the presence of microorganisms can be more or less dangerous, illustrates the potential of this invention. In order to make this possible, the inventors have successfully used the substance cysteine, which the inventors have shown to have unexpectedly strong antimicrobial effect when covalently bound as described and claimed here. Antimicrobial effects have been shown for cysteine analogues and derivatives as well, like N-acetylcysteine and homocysteine.
Polymers or oligomers of ethylene oxide and propylene oxide i.e. poly(ethylene oxide) or poly(ethylene glycol) are readily water soluble and furthermore poly(ethylene oxide) have protein repelling properties, which may be added to the antibacterial function of this invention especially when used in connection with surfaces. Depending on the cysteine component, the following structures are examples of suitable ligands to be used:

In formula (2) the substituents R2, R3 may be hydrogen or alkyl with 1 to 20, preferably from 1 to 12, more preferably 1 to 6 carbon atoms in any combination of R2 and R3 and q may have the same variation as m and p for the methylene constituents of the L segment as previously described i.e. from 1 to 20, preferably from 1 to 12, more preferably from 1 to 6. When q=1 and R2═R3═H the cysteine component becomes a cysteine residue which like the cysteine homologues and derivatives is coupled via its thiol group which contributes with one sulphur to the disulphide bond. In addition to direct alkylation of the cysteine amino group R2 and R3 alkyls may be bound via an amide bond comprising the nitrogen of the cysteine component, e.g. when R2 is hydrogen and R3 is methyl, the cysteine component becomes acetylcysteine.
In formula (3) R2, R3, R4 are alkyl substituents which give a positively charged quartenary amino group. In this case the number of carbon atoms in the alkyl chains of the R2, R3, R4 substituents may vary between 1 and 25, preferably from 1 to 18 in any combination. Further, q may have the same variation as m and p for the methylene constituents of the L segment as previously described i.e. from 1 to 20, preferably from 1 to 12, more preferably from 1 to 6.
Depending on pH, charged ionic groups may also occur as protonized amino groups in (2) and carboxylate-groups in (2) and (3).
The coupling —X— between the subs R1 and the ligand is obtained by chemical reactions between functional groups on R1 and the respective ligand. If R1 has a chemical functionality Y and a ligand functionality Z which upon reaction gives X, the principal coupling reaction where by-products are omitted may be written as:
Depending on the choice of Y and Z and the reaction conditions the X group obtained may be amide, secondary amine, ester, ether, hydrazine, urethane, urea, carbonate and others. A large number of specific and efficient reactions are available which are well established in organic chemistry. The Y, Z and X groups as well as the reactions given here are therefore examples and not limiting for the invention.
TABLE 1(a)when Y = COOHand Z = NH2then X = CONH(b)when Y = COCland Z = NH2then X = CONH(c)when Y = COOHand Z = OHthen X = COO(d)when Y = COCland Z = OHthen X = COO(e)when Y = NH2and Z = CHOthen X = NH(f)when Y = NHNH2and Z = CHOthen X = NHNH(g)when Y = NH2and Z = NCOthen X = NHCONH(h)when Y = NH2and Z = OCOOφNO2then X = NHCOO(i)when Y = NH2and Z = Succinimidyl-then X = NHCO(j)when Y = NH2and Z = Epoxy-then X = NHCH2CH(OH)(k)when Y = OHand Z = NCOthen X = OCONH(l)when Y = OHand Z = Epoxythen X = OCH2CH(OH)(m)when Y = OSO2CH2CF3and Z = NH2then X = CH2NH(n)when Y = OSO2CH2CF3and Z = SHthen X = CH2S(o)when Y = SSand Z = SHthen X = SS(p)when Y = (alkyl)3COKand Z = (alkyl)Brthen X = O(q)when R1 = Au, Agand Z = SHthen X = S(r)when R1 = Au, Agand Z = SSthen X = S(s)when R1 = R1•and Z = CH2═C—then X = CH—C—
The Y and Z groups in examples (a) to (p) may be interchanged between R1 and the ligand to give the same X link although inverted between R1 and the ligand. For example when the functional groups in (a) are interchanged to Y═NH2 and Z═COOH the X link becomes HNOC. In examples (e) and (f) the initially obtained imine generally known as a Schiff base is reduced to the secondary amine link with NaCNBH3. Often intermediate steps are used to increase selectivity and yield. Well known examples are activation of the carboxylic group (Y) in (a) with a carbodiimide and/or N-hydroxysuccinic imide prior to the amide formation with the amino group (Z). Amine groups may be activated with disuccinimidyl carbonate to form urea link with another amine group.
In addition to carboxylic and amino groups hydroxylic groups are also useful in a large number of coupling reactions:                after derivatisation into carbonates giving the Z group in (h) where φ denotes a benzene ring or into tresylated groups as in (m) and (n)        after activation of hydroxylic groups by derivatisation into tosyl- or succinimidyl carbonate groups or treatment with Br2 or CNBr for coupling reactions with nucleophiles like amines and/or thiols.        two hydroxylic groups may be linked together with phosgene to form a carbonate link.        
Comprehensive reviews on coupling chemistry with the reactions mentioned here as well as additional coupling reactions are found in the lyre (Ref. Herman S. et al. J. Bioact. Compat. Pol. 1995, 10, 145-187)
In example (s) in Table 1, R1. symbolizes a solid substrate with free radicals accessible for reaction with an unsaturated group exemplified but not limited to ethylenic, acrylic or metharylic double bonds. By using monomers which have the ligand -L-S—S-(cysteine component) attached to reactive carbon-carbon bonds, oligomeric or polymeric chains may be obtained which are covalently bound to the substrate and which have the ligand as side groups. The concentration of these side groups in the oligomeric or polymeric chains may be controlled by copolymerisation with suitable monomers exemplified by but not limited to acrylic or methacrylic acids or esters or acrylamide. Another route would be to use monomers like maleic acid, maleic anhydride, tiglic acid or allyl amine which readily bind to a free radical providing surface but have strongly reduced chain propagation. The functional groups given by these monomers, i.e. anhydride, carboxyl or amine will therefore become confined to a very thin surface layer on the substrate. By this route each coupling of ligand will occur essentially by terminal attachment directly to functional groups on the surface thus providing a different structure as compared to that obtained when the ligand takes part in graft polymerization.
The coupling reaction with R1—Y may occur according to (4) provided that the Y group reacts selectively with the Z group of the ligand and not with amino or carboxylic groups in the cysteine component.
In cases were the Y group may not react exclusively with the Z group of the ligand represented by:Z-L-S—S-(Cysteine component)  (5)but also with the amino and/or carboxylic group of the cysteine component these groups may, if required, be protested by substitution and esterification respectively. The amino group may be protected by substituents exemplified by tertiary butyloxycarbonyl (t-BuO). This is of course only necessary when the amino group is not alkylated into tertiary or quartenary amines as defined in equations (2) and (3). The carboxyl group of the cysteine component may be protected by methylation. After the coupling reaction between R1—Y and the Z-ligand to obtain the X linked ligand as in equation (4) the t-BuO and the ester-methyl groups may be removed by acid and alkaline hydrolysis respectively and thus restoring the original structure of the Z ligand. With these options the Z-ligand as defined in equation (2)-(4) and by formula (5) and furthermore available with various Z functionalities is a separate item of this invention to be used as a kit component for single step modification of functionalised surfaces. This aspect of the invention also covers the example previously described where the functionality Y of the substrate is a free radical and the functionality Z of the ligand is an unsaturated reactive carbon-carbon bond.
When the functional group Y is bound to a solid substrate the ligand may be synthesized in situ on the substrate surface. This has the advantage that unreacted Y groups as well as by-products are eliminate in intermediary steps.
By this procedure the first step will be to react the R1—Y surface with a compound having the general compositionZ-L-S—S—R5  (6)where L have the same definition as before and where the substituent R5 is easily replaced upon reaction with thiols to give a new disulphide bond with a thiol compound.
The first coupling step may be expressed as:
and the following step:

A common example of R5 is pyridinyl but dansyl is also used. Alternatively the disulphide bond in (6) may belong to a thiosulphate group which will also give a disulphide linkage upon reaction with a thiol.
In addition there is an alternative route for obtaining essentially the same chemical structure as before and which is also within the scope of this invention. In this case a thiol segment or group -L-SH is bound to R1 via the coupling group X and where L and X are defined as before and —SH is a terminal thiol group. This may react exclusively with the thiol group of the cysteine component in the presence of oxidants to form the disulphide link with the cysteine component:

When finally the cysteine component is coupled according to formula (9) an antimicrobial ligand is obtained which is covalently bound to R1, as schematically shown in formula (9).
Surface functionalization of polymeric materials such as plastics, rubber, cellulosics etc. may be achieved by grafting or adsorption of compounds carrying functional grows, such as for example carboxyl or amine. Grafting which gives a covalent link to the substrate requires functionalization of the surface. Compounds which can react with free radicals are grafted during or after activation with UV, electron beam or gamma irradiation or gas plasma. By these methods free radicals may be generated in polymeric substrates which may initiate graft polymerization onto such substrates. These methods of surface modifications for solid polymeric substrates are represented by example (q) in Table 1. In this process the grafting usually involves chain propagation from the substrate surface known as graft polymerization.
Monomers which are frequently used in free radical graft polymerization are acrylic compounds like acrylic acid, methacrylic acid and their esters or acryl amide as well as vinyl pyrrolidone. By graft polymerisation of such monomers containing functional groups e.g. carboxyl, amino, halogens etc, a solid surface may be provided with covalently bound functional groups for covalent attachment of the antimicrobial ligand. A special application of graft polymerisation also comprised by the present invention was previously described where the antimicrobial ligand as depicted in formula (6) could be graft polymerized when Z is a free radical reactive group containing reactive unsaturated carbon-carbon bonds. This would give antimicrobial ligands as side groups in the graft polymerized chains the concentration and location of which may be controlled by graft co-polymerization with suitable vinylic or acrylic monomers. However, as was also previously described, the antimicrobial ligand may be terminally attached directly to the substrate. In this case the functionalisation of the substrate is made in a first step by monomolecular grafting with unsaturated compounds having insignificant chain propagation like maleic anhydride, maleic acid and triflic acid or a self terminating monomer like allyl amine. By this procedure functional groups may be generated on the surface for direct terminal attachment of the antimicrobial ligand by chemical coupling. Also in cases when a vinylic or acrylic ligand in formula (6) will not polymerize e.g. for steric reasons it would attach directly by terminal reaction with free radicals on the substrate.
In cases where the substrate as such is a hydrolyzable plastic material like polyester (PET), polyamide (Nylon™, Nomex™, Kevlar™) or polyacrylate (PMMA) surface functionalization may be obtained by hydrolysis in a basic or acid solution. Polyesters would give carboxylic and hydroxylic groups and polyamides carboxylic and amine groups, which may be used in subsequent modifications by coupling or adsorption.
Metallic substrates like stainless steel may be surface functionalised with carboxylic groups by radiation and plasma treatment. Medical articles like stents are carboxylated by exposure to gas plasma of silane and acrylic acid. Gold and silver surfaces may be grafted by using their reactivity towards thiol and disulphide compounds, which would also carry other groups like carboxyl or amine. Also for metallic substrates free radical grafting of surfaces may be obtained by cathodic polarization of the conductive substrates during exposure to monomers capable of forming covalent links upon reaction with free radicals. The surface grafting is analogous to that for solid polymeric substrates in terms of initiation, propagation and monomers and is also represented by example (s) in the table on table 1.
When surface modification of polymeric substrates is made through adsorption the substrate priming is often made by chemical oxidation, corona treatment or oxidative gas plasma to obtain hydrophilic and ionic groups in the surface layer. One example is the adsorption of polyethylene imine onto polymeric substrates which have been oxidized with permanganate or persulphate. The amino surfaces obtained may be used for chemical coupling reactions as well as adsorption of negatively charged polymers like polyacrylic acid, dextrane sulphate or heparin at suitable pH. Often such polyelectrolytes in their ionically charged states are adsorbed in alternating layers with the properties of interest in the outmost layer. Especially carboxylation of metallic surfaces is often made by adsorption of polyacrylic or polymethacrylic acid. As described above they may then be aminized by chemical coupling or by ionic adsorption of for example polyethylene imine or polyallylamine. Another way to obtain adsorption, which does not need any primary functional on of the substrate, is to use block copolymers having both hydrophobic and hydrophilic blocks or segments which will selectively adsorb to and functionalize the substrate surface. Typical such block copolymers are polyethylene glycol-polypropylene (Pluronics) and polycrylates—polystyrene, polyacrylates—polyethylene, polybutadienes—polystyrene and others which may also contain amino or carboxylic functionalities.
By definition the antibacterial ligand -L-S—S-(cysteine component) of this invention is always covalently bound to a substrate, R1.
However, since the definition of R1 includes organic and polymeric compounds, R1 will also cover polymers which subsequently are capable of binding to a solid substrate by covalent binding or adsorption.
The options for covalent binding of the antibacterial agent of this invention onto a solid substrate are emphasized by the definition of R1, comprising that attachment of the antimicrobial agent to a solid substrate by adsorption. In this case R1 is a soluble substrate, exemplified by e.g. ionizable polymers like polyethylene imine or polyacrylic acid or block copolymers with hydrophobic/hydrophilic blocks like polyethylene glycol-polypropylene glycol or polyacrylates in block copolymers with polystyrene, polyethylene and others. The cysteine component is covalently bound to a soluble substrate which in a further step is immobilized on a solid substrate as described here.
Thus, the surface modifications as well as the subsequent chemical coupling or adsorption used to attach the antibacterial agents may be performed by many different routes. In addition the substrates may be organic or inorganic materials comprising synthetic or natural polymers as well as metals and minerals. Therefore the methods, chemical reactions and substrates, which are presented here and in the examples below, are only descriptive and not limiting for obtaining the antibacterial agents and surfaces covered by the invention.
The surface concentration of the agent of the invention, such as L-cysteine, is in the interval from 10−11 from 10−4 mole/cm2, and preferably in the interval from 10−9 to 10−5 mole/cm2.
In order to obtain inhibition of clinically or technically important microorganisms a 100-folded inhibition is preferably achieved in accordance with the invention with respect to adherent viable bacteria that can be released with the assay, staring with a large exposure (titer of 400 000 cfu/ml in the staring culture). This may partly be dependent on the specific organism and the degree/titer of exposure. The tested conditions vastly exceed what can be expected in the actual clinical situation.
Examples of microorganisms, for which the invention may be used to prevent growth and/or proliferation of, are anaerobic and aeorbic bacteria that encompass both different Gram-positive bacteria chosen from, but not limited to, different species of Staphylococci such as S. aureus, S. epidermides and other coagulase-negative staphylococci, S. saphrophyticus, Enterococcus spp, Nesseriae (Meningococci Gonococci), Streptococci (Viridans, hemolytic and non-hemolytic, group B and D, S. pneumoniae), Chlostridia (perfringens, botulinum), Bacillus megaterium, as well as different Gram-negative species chosen from, but not limited to, different Enterobacter spp, Escherichia coli, Kiebsiella spp, Proteus, Campylobacter, Yersinia, Shigella, Salmonella, Hemophilus (influenza), Barteriodes (fragilis, bivius), Pseudomonas (aeruginosa, cepacia), Legionella (pneumophilia). Also included are different mycoplasma species and candida species and different fungi. Preferred examples of bacteria are the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, or the Gram-positive bacterium Bacillus megaterium. 
The invention can be used to prevent or inhibit growth of microorganisms on surfaces of different applications that can cause a problem due to colonisation or infection. It has here been shown to be effective against both Gram positive and Gram negative bacteria (the Gram negative bacterium Escherichia coli, or the Gram positive bacterium Staphylococcus aureus and Bacillus megaterium). Several different microorganisms have been described in relation to catheter colonisation and infection in the health care sector and hospital environment. These microorganisms include, but are not limited to, Gram positive and Gram negative bacteria listed below. Also different fungi are a frequent problem, especially in immuno-compromised patients (undergoing transplantation, or otherwise immunosuppressive therapy etc). The invention can be utilised where infection, colonisation or biofilm formation on artificial devices (catheters, trachiostomi tubes etc) can be a problem in health care. Examples of microorganisms known or described to be catheter borne, and against which the invention can be used, are (but not limited to): Staphylococci spp (such as S. aureus, S. epidermides and other coagulase-negative Staphylococci like S. saprophyticus); Streptococci spp (viridans, hemolytic and non-hemolytic, group B and D, S. pneumoniae; Enterococcus spp, S. facealis; Chlostidia (perfingens, botulinm); Different Enterobacter spp, like Escherichia coli, Klebsiella spp (pneumonia), Enterobacter cloace, aerogenes, Proteus, (mirabilis), Campylobacter, Yersinia, Shigella, Salmonella, Hemophilus (influenza), Neisseriae (meningococcus and gonococcus), Bacteroides (bacteroides Spp. and fasobacterium), Pseudomonas (aeruginosa, cepacia), Legioneila (pneumophilia), Sertatia marcenens, Acinetobacter spp, Morganella morganii, Stenotrophomonas, Citrobacter spp, Corynebacterium spp, Burkholder Cepafia, Acinetobacter spp; Different Mycoplasma species (M. avian and other); and also fingi such as Candida spp, C. tropicales, C. parapsilosis, Cryptococcus neoformans, Aspergillus fumigatus, Tricosporun, Blastoschizomyces, Stenotrophomonas maltophilia, Malassezia, Bukholderia cepafa, Aspergillus. 
In many applications of the present invention, the substrate is apart of a device, an apparatus and/or a surface chosen from (a) medical devices, such as extracorporeal medical devices, which are applicable at the exterior of the human or animal body or infracorporeal medical devices, which are applicable in the interior of the human or animal body, (b) grocery devices, and (c) other devices. The examples of applications listed below are only intended to demonstrate the potential of the invention without in any way being limiting.
Medical devices (a) comprise applications chosen from:                artificial skin or cove for burning wounds        dialysis (tubings from an to the dialysis device)        ear drainage (drainage from a cavity, wound or abscess or within the interior of the ear)        ear implants (implant within the interior of the ear)        hearing aid device (interiorally placed hearing device)        heart and lung machine tubings (tubings from and to the heart and lung machine device)        hydrocephalus drainage (drainage from the brain region/ventricles)        syringe (disposable syringes)        stomis (stomi devices)        suture materials (suture devices)        wound care (wound care devices, such as plaster).        catheters (disposable and permanent catheter devices, e.g. central venous catheters (CVC), peripheral venous catheters (PVC), peripherally inserted central catheters, urinary catheters, and peritoneal catheters)        dental products (products implanted in the mouth region)        implants in the body (bones, pro paradontit (products implanted in the mouth region))        insulin pump (tubings from and to the insulin pump)        nerve guidelines (guiding devices for nerves)        pacemaker (pacemaker and its surrounding devices)        postoperative drainage (drainage devices subsequent to surgery)        drainage from regions and/or organs and cavities within the human body (abscess, nephrostomi and similar)        intracorporeal/intraluminal stents (stents used to keep different lumen open, for example in the vascular system and vessels, in organs and tissue, in the intestinal system, bile ducts etc)        tubing or equipment used for parenteral administration of liquids, solutions, infusions, drug delivery.        
Grocery devices (b) comprise applications chosen from:                contact for fresh food or subsumes or devices used in food processing (surfaces that may be in contact with bacterial sources)        drug packages (to keep the opening free of bacterias) packaging for sensitive drugs)        milking devices (devices subjected to bacterial sources during milking operations)        sprinkler devices (sprinkler devices and other wax transporting devices that may be colonised by microorganisms, such as mouthpiece in grocery store)        roller steel within fishing industry (rollers used in the fish industry in order to enhance the production of fish products).        
Miscellaneous devices (c) comprise applications chosen from:                contact lenses (ordinary contact lenses)        intraocular lenses        cosmetic package products (packages for different cosmetic products)        water tanks (water tanks that contain tap water or recirculating water)        water pipes (pipes that transport tap water or recirculating water)        air conditioning, air and water cooling devices,        other devices for storage of products or materials where bacterial growth on surfaces of storage material is undesired.        
In all these application, the antimicrobial agent of the invention is coupled to a surface of the device, as discussed above, in order to confer the antimicrobial effect.
In a preferred embodiment, the antimicrobial agent is coupled to a catheter surface, thereby providing a catheter being able to prevent growth and/or proliferation of microorganisms. Normally the inside and/or the outside surface of the catheter is coated with the agent of the invention. The coating process catheter surface may further be incorporated during or after the extrusion of the catheter, or as a separate step, prior to or after the assembly of the specific catheter. For treated catheter samples the microbial effect has been shown to remain after several years of storage under ambient conditions. Catheters that can be used in the invention are provided from commercial suppliers of catheter channels, e.g. Rehau, Habia, Vygon, Teknofluor, Optinova, Baxter etc.
In yet another aspect, the invention refers to a kit of parts for use in treating a surface with an antimicrobial agent, comprising, in separate compartments, (a) a precursor to the antimicrobial agent of claim 1, the precursor having the formula:Z-L-S—S-(cysteine component)wherein L, m, and cysteine component is as defined above, and Z is a ligand functionality as defined above that can react with a chemical compound Y as defined above to give the chemical compound X as defined above, and (b) necessary reagents in order to covalently bind the precursor to a surface, wherein the kit further comprises instructions for using the kit Necessary reagents may comprise any reagents that are necessary for performing a coupling reaction of Y and Z to obtain X (as outlined above), and would depend on the specific identity of Y and Z. The person skilled in the art would know what reagents that would be necessary in each situation.
Hereby, a kit is provided which can be used to treat any desired surface with the antimicrobial agent of the invention in order to give the surface antimicrobial properties.
The invention will now be further illustrated by way of examples. These examples are of illustrative purpose only, and shall not be regarded as limiting the scope of the invention in any way.